Lysosomal Disorders Drive Susceptibility to Tuberculosis by Compromising Macrophage Migration.

A zebrafish genetic screen for determinants of susceptibility to Mycobacterium marinum identified a hypersusceptible mutant deficient in lysosomal cysteine cathepsins that manifests hallmarks of human lysosomal storage diseases. Under homeostatic conditions, mutant macrophages accumulate undigested lysosomal material, which disrupts endocytic recycling and impairs their migration to, and thus engulfment of, dying cells. This causes a buildup of unengulfed cell debris. During mycobacterial infection, macrophages with lysosomal storage cannot migrate toward infected macrophages undergoing apoptosis in the tuberculous granuloma. The unengulfed apoptotic macrophages undergo secondary necrosis, causing granuloma breakdown and increased mycobacterial growth. Macrophage lysosomal storage similarly impairs migration to newly infecting mycobacteria. This phenotype is recapitulated in human smokers, who are at increased risk for tuberculosis. A majority of their alveolar macrophages exhibit lysosomal accumulations of tobacco smoke particulates and do not migrate to Mycobacterium tuberculosis. The incapacitation of highly microbicidal first-responding macrophages may contribute to smokers' susceptibility to tuberculosis.This work was supported by the National Institutes of Health (R37AI054503, LR, R01NS082567, CBM, 5F30HL110455, RB, 1DP2-OD008614, DMT), the Wellcome Trust (LR), the National Institute of Health Research Cambridge Biomedical Research Centre (LR), the Health Research Board of Ireland (HRA_POR/2013/387, MO’S and CSA/2012/16, JK), and The Royal City of Dublin Hospital Trust (Grant 146, JK).This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.cell.2016.02.034

Mycobacterial Acid Tolerance Enables Phagolysosomal Survival and Establishment of Tuberculous Infection In Vivo.

The blockade of phagolysosomal fusion is considered a critical mycobacterial strategy to survive in macrophages. However, viable mycobacteria have been observed in phagolysosomes during infection of cultured macrophages, and mycobacteria have the virulence determinant MarP, which confers acid resistance in vitro. Here we show in mice and zebrafish that innate macrophages overcome mycobacterial lysosomal avoidance strategies to rapidly deliver a substantial proportion of infecting bacteria to phagolysosomes. Exploiting the optical transparency of the zebrafish, we tracked the fates of individual mycobacteria delivered to phagosomes versus phagolysosomes and discovered that bacteria survive and grow in phagolysosomes, though growth is slower. MarP is required specifically for phagolysosomal survival, making it an important determinant for the establishment of mycobacterial infection in their hosts. Our work suggests that if pathogenic mycobacteria fail to prevent lysosomal trafficking, they tolerate the resulting acidic environment of the phagolysosome to establish infection.National Institutes of Health (Grant IDs: R37AI054503, R01 AI076327, 5T32HD007233, 5F30HL110455), Wellcome Trust, National Institute of Health Research Cambridge Biomedical Research CentreThis is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.chom.2016.07.00

Multifaceted roles of the lysosome in the establishment and expansion of tuberculous infection

Thesis (Ph.D.)--University of Washington, 2016-04Infection with Mycobacterium tuberculosis, the causative agent of TB, begins with the deposition of bacteria into the lung alveolus where they establish infection in host macrophages. M. tuberculosis dwells within host macrophages for much of its life cycle, until it again becomes extracellular prior to transmission into a new host. M. tuberculosis’ use of the macrophage as an expansion niche is surprising because these immune cells are normally tasked with the elimination of invading bacteria by trafficking them to degradative lysosomal compartments. Using the optically transparent and genetically tractable zebrafish model of mycobacterial infection with M. marinum, I have evaluated the diverse roles played by the macrophage lysosome at two stages of infection: first, during the initial interaction between infecting mycobacteria and first-responder macrophages; and second, during the stage of infection when bacteria are released from the macrophage and grow extracellularly. When newly infecting bacteria are phagocytosed by macrophages, their survival within these broadly microbicidal cells is thought to depend upon their ability to block phagosome-lysosome fusion and subsequent acidification. This notion persists despite multiple studies in cultured macrophages, spanning several decades, reporting a substantial proportion of viable mycobacteria in phagolysosomes. This work shows that while lysosomal trafficking in the macrophage can slow bacterial replication, it does not kill the pathogen. For its part, the mycobacterium survives within the macrophage lysosome through the action of a highly conserved virulence factor that mediates acid tolerance in vitro. A concurrent forward genetic screen identified a zebrafish mutant that is hypersusceptible to M. marinum infection and manifests hallmarks of human lysosomal storage diseases. The mutant is deficient in the lysosomal hydrolases Cathepsin B and L, although its hypersusceptibility does not stem from defective intramacrophage restriction of mycobacterial growth. Rather the mutant’s hypersusceptibility is attributed to defective macrophage migration owing to accumulated undegraded cell products that accumulate through normal homeostatic macrophage function. Similarly, macrophage migration defects underlie hypersusceptibility to mycobacterial infection following disruption of the zebrafish orthologs of genes implicated in human lysosomal storage disorders. All of these migration defects share a common mechanism in disrupted endocytic recycling caused by the lysosomal accumulation of undigestible cargo. Macrophage immotility resulting from lysosomal storage defects causes hypersusceptibility by compromising migration to dying infected cells in the granuloma, resulting in its breakdown with extracellular release of bacteria. Lysosomal storage defects also disrupt macrophage participation in the earliest stage of infection when resident macrophages migrate to and phagocytose newly arriving mycobacteria. The alveolar (lung resident) macrophages of human smokers display lysosomal accumulations of tobacco smoke and fail to migrate to M. tuberculosis. This may explain the increased susceptibility of human smokers to TB. This work defines the limited protection afforded to the host by lysosomal trafficking in the restriction of intramacrophage mycobacterial replication, and elucidates the consequences of lysosomal dysfunction, which results in host susceptibility through a completely distinct mechanism of macrophage incapacitation following the lysosomal accumulation of indigestible cargo

Therapeutic potential of exosomes derived from mesenchymal stem cells for treatment of systemic lupus erythematosus

Abstract Autoimmune diseases are caused by an imbalance in the immune system, producing autoantibodies that cause inflammation leading to tissue damage and organ dysfunction. Systemic Lupus Erythematosus (SLE) is one of the most common autoimmune diseases and a major contributor to patient morbidity and mortality. Although many drugs manage the disease, curative therapy remains elusive, and current treatment regimens have substantial side effects. Recently, the therapeutic potential of exosomes has been extensively studied, and novel evidence has been demonstrated. A direct relationship between exosome contents and their ability to regulate the immune system, inflammation, and angiogenesis. The unique properties of extracellular vesicles, such as biomolecule transportation, biodegradability, and stability, make exosomes a promising treatment candidate for autoimmune diseases, particularly SLE. This review summarizes the structural features of exosomes, the isolation/purification/quantification method, their origin, effect, immune regulation, a critical consideration for selecting an appropriate source, and their therapeutic mechanisms in SLE

Precision Delivery of Human Bone Marrow-Derived Mesenchymal Stem Cells Into the Pancreas Via Intra-arterial Injection Prevents the Onset of Diabetes

Mesenchymal stem cells (MSCs) are a promising therapy to potentially treat diabetes given their potent anti-inflammatory and immune-modulatory properties. While these regenerative cells have shown considerable promise in cell culture, their clinical translation has been challenging. In part, this can be attributed to these cells not reaching the pancreas to exert their regenerative effects following conventional intravenous (IV) injection, with the majority of cells being trapped in the lungs in the pulmonary first-pass effect. In the present study, we will therefore examine whether direct delivery of MSCs to the pancreas via an intra-arterial (IA) injection can improve their therapeutic efficacy. Using a mouse model, in which repetitive low doses of STZ induced a gentle, but progressive, hyperglycemia, we tested bone marrow-derived MSCs (BM-MSCs) which we have shown are enriched with pro-angiogenic and immunomodulatory factors. In cell culture studies, BM-MSCs were shown to preserve islet viability and function following exposure to proinflammatory cytokines (IFN-γ, IL-1β, and TNF-α) through an increase in pAkt. When tested in our animal model, mice receiving IV BM-MSCs were not able to mitigate the effects of STZ, however those which received the same dose and batch of cells via IA injection were able to maintain basal and dynamic glycemic control, to similar levels as seen in healthy control animals, over 10 days. This study shows the importance of considering precision delivery approaches to ensure cell-based therapies reach their intended targets to enable them to exert their therapeutic effects

glo-3, a Novel Caenorhabditis elegans Gene, Is Required for Lysosome-Related Organelle Biogenesis

Gut granules are specialized lysosome-related organelles that act as sites of fat storage in Caenorhabditis elegans intestinal cells. We identified mutations in a gene, glo-3, that functions in the formation of embryonic gut granules. Some glo-3(−) alleles displayed a complete loss of embryonic gut granules, while other glo-3(−) alleles had reduced numbers of gut granules. A subset of glo-3 alleles led to mislocalization of gut granule contents into the intestinal lumen, consistent with a defect in intracellular trafficking. glo-3(−) embryos lacking gut granules developed into adults containing gut granules, indicating that glo-3(+) function may be differentially required during development. We find that glo-3(+) acts in parallel with or downstream of the AP-3 complex and the PGP-2 ABC transporter in gut granule biogenesis. glo-3 encodes a predicted membrane-associated protein that lacks obvious sequence homologs outside of nematodes. glo-3 expression initiates in embryonic intestinal precursors and persists almost exclusively in intestinal cells through adulthood. GLO-3∷GFP localizes to the gut granule membrane, suggesting it could play a direct role in the trafficking events at the gut granule. smg-1(−) suppression of glo-3(−) nonsense alleles indicates that the C-terminal half of GLO-3, predicted to be present in the cytoplasm, is not necessary for gut granule formation. Our studies identify GLO-3 as a novel player in the formation of lysosome-related organelles